Methods for quantitation of insulin and C-peptide

Abstract

Methods are described for diagnosing or prognosing insulin resistance in diabetic and pre-diabetic patients, the method comprising determining the amount of insulin and C-peptide in a sample. Provided herein are mass spectrometric methods for detecting and quantifying insulin and C-peptide in a biological sample utilizing enrichment and/or purification methods coupled with tandem mass spectrometric or high resolution/high accuracy mass spectrometric techniques.

That which is claimed is: 
     
       1. A method for measuring insulin resistance in diabetic or pre-diabetic patients by mass spectrometry, the method comprising:
 (a) purifying a sample comprising insulin and C-peptide by liquid chromatography; 
 (b) ionizing the insulin and the C-peptide by an ionization source under conditions suitable to generate one or more insulin and C-peptide ions detectable by mass spectrometry; and 
 (c) determining an amount of the one or more insulin and C-peptide ions by mass spectrometry; 
 (d) determining an amount of the insulin and the C-peptide in the sample from the amount of the one or more insulin and C-peptide ions; and 
 (d) determining an insulin resistance score (RS) and/or a probability of developing insulin resistance (P(IR)) from the amount of the insulin and the C-peptide in the sample, wherein insulin resistance in diabetic or pre-diabetic patients is measured from the RS and/or the P(IR); 
 wherein 
 
       
         
           
             
               RS 
               = 
               
                 
                   ( 
                   
                     Insulin 
                     × 
                     0.0295 
                   
                   ) 
                 
                 + 
                 
                   ( 
                   
                     C 
                     ⁢ 
                     ‐ 
                     ⁢ 
                     
                       peptide 
                       × 
                       0.00372 
                     
                   
                   ) 
                 
               
             
           
         
         
           
             
               
                 P 
                 ⁡ 
                 ( 
                 IR 
                 ) 
               
               = 
               
                 
                   
                     e 
                     
                       
                         - 
                         4.5046 
                       
                       + 
                       
                         1.0001 
                         × 
                         RS 
                       
                     
                   
                   
                     1 
                     + 
                     
                       e 
                       
                         
                           - 
                           4.5046 
                         
                         + 
                         
                           1.0001 
                           × 
                           RS 
                         
                       
                     
                   
                 
                 . 
               
             
           
         
       
     
     
       2. The method of  claim 1 , wherein the method further comprises measuring creatinine levels. 
     
     
       3. The method of  claim 1 , wherein the method further comprises measuring body mass index (BMI). 
     
     
       4. The method of  claim 1 , wherein the method further comprises measuring triglyceride (TG) levels. 
     
     
       5. The method of  claim 1 , wherein the method further comprises measuring high density lipoprotein C (HDL-C) levels. 
     
     
       6. The method of  claim 1 , wherein the method further comprises measuring BMI, TG, and HDL-C levels. 
     
     
       7. The method of  claim 1 , wherein the sample comprises a plasma or serum sample. 
     
     
       8. The method of  claim 1 , wherein the ionization source is an electrospray (ESI) ionization source. 
     
     
       9. The method of  claim 1 , wherein the sample is subjected to acidic conditions prior to mass spectrometry. 
     
     
       10. The method of  claim 9 , wherein subjecting the sample to acidic conditions comprises subjecting the sample to formic acid. 
     
     
       11. The method of  claim 1 , wherein the sample is subjected to basic conditions prior to mass spectrometry. 
     
     
       12. The method of  claim 11 , wherein subjecting the sample to basic conditions comprises subjecting the sample to trizma and/or ethanol. 
     
     
       13. The method of  claim 1 , wherein the one or more ions comprise an insulin precursor ion has a mass to charge ratio (m/z) of 968.9±0.5. 
     
     
       14. The method of  claim 1 , wherein the one or more ions comprise one or more insulin fragment ions selected from the group consisting of ions with m/z of 136.0±0.5, 226.1±0.5, and 345.2±0.5. 
     
     
       15. The method of  claim 1 , wherein the one or more ions comprise a C-peptide precursor ion has a mass to charge ratio (m/z) of 1007.7±0.5. 
     
     
       16. The method of  claim 1 , wherein the one or more ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m/z of 533.3±0.5, 646.4±0.5, and 927.5±0.5. 
     
     
       17. The method of  claim 1 , wherein the sample is delipidated prior to quantitation by mass spectrometry. 
     
     
       18. The method of  claim 1 , wherein liquid chromatography comprises high performance liquid chromatography (HPLC) or high turbulence liquid chromatograph (HTLC). 
     
     
       19. The method of  claim 1 , wherein the purifying further comprises subjecting a sample to solid phase extraction (SPE). 
     
     
       20. The method of  claim 1 , wherein the mass spectrometry is tandem mass spectrometry, high resolution mass spectrometry, or high resolution/high accuracy mass spectrometry. 
     
     
       21. The method of  claim 1 , wherein ionization is in positive ion mode. 
     
     
       22. The method of  claim 1 , wherein an internal standard for insulin and an internal standard for C-peptide are added to the sample. 
     
     
       23. The method of  claim 22 , wherein the internal standard for insulin is bovine insulin. 
     
     
       24. The method of  claim 23 , wherein the bovine insulin comprises a precursor ion with a mass to charge ratio (m/z) of 956.8±0.5 and fragment ions selected from the group consisting of ions with a m/z of 136.0±0.5, 226.1±0.5, and 315.2±0.5. 
     
     
       25. The method of  claim 24 , wherein the internal standard for C-peptide is a C-peptide heavy internal standard. 
     
     
       26. The method of  claim 25 , wherein the C-peptide heavy internal standard comprises a precursor ion with a mass to charge ratio (m/z) of 1009.5±0.5 and fragment ions selected from the group consisting of ions with m/z of 540.3±0.5, 653.4±0.5, and 934.5±0.5. 
     
     
       27. The method of  claim 1 , wherein the amount of the insulin and the C-peptide in the sample is used to determine a ratio of insulin to C-peptide. 
     
     
       28. The method of  claim 1  further comprising subjecting a sample to an enrichment process to obtain a fraction enriched in insulin and C-peptide. 
     
     
       29. The method of  claim 28 , wherein the enrichment process comprises an immunocapture of insulin and C-peptide. 
     
     
       30. The method of  claim 29 , wherein the immunocapture comprises using anti-insulin antibodies and anti-C-peptide antibodies. 
     
     
       31. The method of  claim 30 , wherein the anti-insulin antibodies and the anti-C-peptide antibodies are monoclonal antibodies. 
     
     
       32. The method of  claim 31 , wherein the monoclonal antibodies are monoclonal IgG antibodies. 
     
     
       33. The method of  claim 30 , wherein the anti-insulin antibodies and the anti-C-peptide antibodies are immobilized on magnetic beads. 
     
     
       34. The method of  claim 33 , wherein the insulin and the C-peptide immunocaptured on the magnetic beads are washed and eluted. 
     
     
       35. The method of  claim 1 , wherein insulin resistance is diagnosed if the amount of C-peptide is greater than or equal to 2.4 ng/mL. 
     
     
       36. The method of  claim 1 , wherein insulin resistance is diagnosed if the amount of insulin is greater than or equal to 15 μIU/mL. 
     
     
       37. The method of  claim 1 , wherein insulin resistance is diagnosed if the amount of C-peptide is greater than or equal to 2.4 ng/mL and the amount of insulin is greater than or equal to 15 μIU/mL.